**7. Concluding Remarks**

The hypothesis that parasites influence the population size or geographical dispersion of their host is opposed by a more acceptable hypothesis arguing that successful or welladapted parasites evolve to be harmless to their host. Although virus-like particles and viruses were first detected in *Leishmania* parasites some decades ago, the impact of this interaction and the diversity of these endosymbionts have recently drawn considerable attention, mainly due to the virulence trade-off experimentally demonstrated in the context of *Leishmania* (*Viannia*) *guyanensis* and LRV1. However, the theory regarding the evolution of interactions among different endosymbiotic viruses and *Leishmania* spp. is still in its emerging stages. Recent studies have reported the discovery of several viruses in trypanosomatids, indicating the existence of unknown viral diversity, which needs to be further investigated and can provide important evolutionary information. At least two

virus families have already been described as *Leishmania* spp. endosymbionts, but we still do not know if these viruses occur only in *Leishmania* spp. or if they can be detected elsewhere, such as in the invertebrate host of *Leishmania* spp. It is plausible to assume a dynamic symbiotic relationship in this long-term interaction between LRV or LBV and *Leishmania* spp., but the influence of either LRV or LBV on *Leishmania* biology is not yet clear. At least in some circumstances, it seems that this interaction causes a stressful condition, promoting increased tolerance of *Leishmania* spp. to some environments and augmenting its replication rate. It is a fact that both viruses influence leishmaniasis pathogenesis, but it is still unclear whether this is a consequence of the vertebrate host response to the virus living in *Leishmania* spp. cytoplasm or a biological response of *Leishmania* spp. to the endosymbiotic viruses. The impact of a "parasitized parasite" in the initial moments of a natural infection is also an aspect that deserves attention. The phenotype of higher pathogenicity linked to *Leishmania* spp. bearing viruses might be linked to an increased evolutionary fitness might be considered to signal that viral acquisition was beneficial to the parasite. However, there is also the possibility of better fitness for those organisms that are less pathogenic, which could have the chance to produce asymptomatic infections, to be maintained longer in the vertebrate host, and to be dispersed to new environments.

The screening of viruses in *Leishmania* spp. is still limited to a few studies, but so far, the evidence has indicated that LRV1 is restricted to the American continent and associated with *Leishmania* (*Viannia*) species and that LRV2 is linked to the Old World and hosted by *Leishmania* (*Leishmania*). LBV was detected only in *L*. *martiniquensis*, a species belonging to a subgenus not closely related to *L*. (*Viannia*) or *L*. (*Leishmania*). For both LRV1 and LRV2, there were different genotypes and correlations with the parasitized *Leishmania* species. The consequence of *Leishmania*-LRV or *Leishmania*-LBV coevolution was probably dependent on coevolutionary dynamics, involving (i) fluctuating selection affecting the frequency of some genotypes, especially those linked to resistance and infectivity [92] or fluctuations in the ranges of resistance and infectivity [93], and (ii) antagonist coevolution turning towards either increasing infectivity, resistance, or both. There is an important imbroglio of evolution and ecology linked to the relationship between *Leishmania* spp. and LRV or LBV, these interactions providing a direct impact on the evolutionary route.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/genes12050657/s1, Supplementary Data 1: LRV1 detection in *Leishmania* spp. Table S1: Strains of *L*. (*Viannia*) positive for LRV1. Supplementary Data 2: Growth curve of *Leishmania guyanensis* strains infected or not with the *Leishmania* RNA virus in single or mixed culture. Figure S1: Growth curve of *Leishmania guyanensis* strains infected or not with the *Leishmania* RNA virus in single or mixed culture.

**Author Contributions:** L.M.C. and E.C., conceptualization, writing and design of the figures; M.C.B. and E.C., scientific critical overview and constant writing revision; C.M.-S., K.C., G.P.d.S., and B.D.d.C., contributed with unpublished data; C.M.-S. and L.d.O.R.P., writing—review and editing. All authors contributed to the final critical review of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the Instituto Nacional de Ciência e Tecnologia, INCT-EpiAmo. Coordenação de Aperfeiçoamento de Pessoal de Nível Superior–Brasil (CAPES)–Finance Code 001; Programa PRINT FIOCRUZ-CAPES. CNPq (Researcher Fellow, 302622/2017-9). Faperj (CNE, E26- 202.569/2019). IOC-Fiotec (PAEF LPL) Oswaldo Cruz Foundation.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.
